Somatic mutations in isocitrate dehydrogenase 1 (IDH1) and IDH2 occur frequently in a number of cancers, including glioma, acute myeloid leukemia, and chondrosarcoma. These mutations help initiate tumorigenesis via neomorphic production of the (D)2-hydroxyglutarate oncometabolite but also compromise the activity of important metabolic pathways. Our preliminary studies indicate that a critical function of the tricarboxylic acid (TCA) cycle that supports tumor growth under hypoxia is dysfunctional in IDH1 mutant tumors, and this defect can be targeted to specifically limit the growth of cancer cells with this genotype. In this project we will apply systems biology approaches and novel analytical tools to identify additional metabolic liabilities in IDH mutant cancer cells. These methods will provide crucial new insights into how mitochondrial metabolism and the redox state of tumor cells are regulated in cancer cells generally and IDH mutant lines in particular. We hypothesize that TCA metabolism and cofactor-dependent redox pathways are critically perturbed in IDH mutant tumors, and these defects can be exploited to selectively mitigate tumor growth and survival. In Aim 1 we will characterize the reprogramming of central carbon metabolism by oncogenic IDH1 and IDH2 mutations using 13C MFA. In Aim 2 we will identify critical metabolic sources of cytosolic and mitochondrial NADPH in cells with oncogenic IDH1 and IDH2. Importantly, the metabolism of tumors within the in vivo microenvironment may differ significantly from that observed in vitro. In Aim 3 we will validate our metabolic findings and target efficacy in xenograft models. Collectively, the information gained from our metabolic systems biology approach will be used to develop and validate therapeutic strategies that exploit these metabolic defects in cancer with IDH mutations.